Pharmaceutical compositions comprising polymorphic forms alpha, beta, and gamma of rifaximin

ABSTRACT

Crystalline polymorphous forms of rifaximin (INN), referred to as rifaximin α and rifaximin β, and a poorly crystalline form referred to as rifaximin γ, useful in the production of medicaments containing rifaximin for oral and topical use and obtained by means of a crystallization process carried out by hot-dissolving the raw rifaximin in ethyl alcohol and by causing the crystallization of the product by addition of water at a fixed temperature and for a fixed period of time, followed by a drying under controlled conditions until reaching a precise water content in the end product, are the object of the invention.

This application is a continuation of U.S. application Ser. No.13/041,347, filed Mar. 4, 2011, now U.S. Pat. No. 8,158,644, issued onApr. 17, 2012, which in turn is a continuation of U.S. application Ser.No. 12/119,622, filed May 13, 2008, now U.S. Pat. No. 7,906,542, issuedon Mar. 15, 2011, which in turn is a continuation-in-part of applicationU.S. application Ser. No. 11/873,841, filed on Oct. 17, 2007, now U.S.Pat. No. 7,915,275, issued on Mar. 29, 2011, which is a continuation-inpart of U.S. application Ser. No. 11/135,651, filed on May 24, 2005, nowabandoned, which is a continuation-in-part of PCT/EP04/12490, filed onNov. 4, 2004, which claims priority to Italian application No.MI2003A002144 filed Nov. 7, 2003, all of which are incorporated byreference herein in their entirety, including any drawings.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to rifaximin polymorphic forms α, β and γ,the processes for their preparation and the use thereof in themanufacture of medicinal preparations.

2. Description of the Related Art

Rifaximin (INN; see The Merck Index, XIII Ed., 8304) is an antibioticbelonging to the rifamycin class, namely a pyrido-imidazo rifamycindescribed and claimed in Italian Patent IT 1154655, while EP 0161534discloses and claims a process for its production starting fromrifamycin O (The Merck Index, XIII Ed., 8301).

Both these patents generically describe the purification of rifaximin bycrystallization in suitable solvents or solvent systems and summarilyshow in some examples that the resulting product can be crystallizedfrom the 7:3 mixture of ethyl alcohol/water and dried both underatmospheric pressure and under vacuum. Neither information concerningthe experimental conditions of crystallization and drying, nor anydistinctive crystallographic characteristic of the obtained product arereported.

The presence of different polymorphs had not been ascertained andtherefore the experimental conditions described in both patents had beendeveloped with the aim of obtaining a homogeneous product havingsuitable purity from the chemical point of view, apart from thecrystallographic aspects of the product itself.

It has now been unexpectedly found that some polymorphic forms ofrifaximin exist whose formation depends on the solvent as well as on theconditions of time and temperature at which both crystallization anddrying are carried out.

SUMMARY OF THE INVENTION

In one embodiment, a pharmaceutical composition is provided comprisingrifaximin in polymorphic form α. In one embodiment, the C_(max) is about2 ng/ml. In a further embodiment, the C_(max) is about 2.6 ng/ml. In oneembodiment, the t_(max) is about 9 hours. In a further embodiment, thet_(max) is about 9.5 hours. In other embodiments, the AUC_(0-24 h) isabout 13 ng·h/ml.

In another embodiment, the pharmaceutical composition providedcomprising rifaximin in polymorphic form α has a water content of frombetween about 0% to about 3%. In one embodiment, the C_(max) is about 2ng/ml. In a further embodiment, the C_(max) is about 2.6 ng/ml. In oneembodiment, the t_(max) is about 9 hours. In a further embodiment, thet_(max) is about 9.5 hours. In other embodiments, the AUC_(0-24 h) isabout 13 ng·h/ml.

In other embodiments, a pharmaceutical composition is providedcomprising rifaximin in polymorphic form β. In one embodiment, theC_(max) is about 1 ng/ml. In a further embodiment, the C_(max) is about1.1 ng/ml. In one embodiment, the t_(max) is about 4 hours. In a furtherembodiment, the t_(max) is about 9.5 hours. In other embodiments, theAUC_(0-24 h) is about 11 ng·h/ml. In one embodiment, the intrinsicdissolution rate of the rifaximin is about 0.01 mg/min/cm².

In another embodiment, the pharmaceutical composition providedcomprising rifaximin in polymorphic form β has a water content of frombetween greater than about 4.5%. In one embodiment, the C_(max) is about1 ng/ml. In a further embodiment, the C_(max) is about 1.1 ng/ml. In oneembodiment, the t_(max) is about 4 hours. In a further embodiment, thet_(max) is about 9.5 hours. In other embodiments, the AUC_(0-24 h) isabout 11 ng·h/ml. In one embodiment, the intrinsic dissolution rate isabout 0.01 mg/min/cm².

In other embodiments, a pharmaceutical composition is providedcomprising rifaximin in polymorphic form γ. In one embodiment, theC_(max) is about 670 ng/ml. In a further embodiment, the C_(max) isabout 668 ng/ml. In one embodiment, the t_(max) is about 2 hours. In afurther embodiment, the t_(max) is about 2.3 hours. In otherembodiments, the AUC_(0-24 h) is about 4000 ng·h/ml.

In another embodiment, the pharmaceutical composition providedcomprising rifaximin in polymorphic form γ has a water content of frombetween about 1% to about 2%. In one embodiment, the C_(max) is about670 ng/ml. In a further embodiment, the C_(max) is about 668 ng/ml. Inone embodiment, the t_(max) is about 2 hours. In a further embodiment,the t_(max) is about 2.3 hours. In other embodiments, the AUC_(0-24 h)is about 4000 ng·h/ml.

Another embodiment of the invention provides a pharmaceuticalcomposition comprising one or more of a form α, a form β, and a form γpolymorph of rifaximin and a pharmaceutically acceptable carrier. In oneembodiment, the pharmaceutical composition further comprises excipients.In a further embodiment, the excipients are one or more of a dilutingagent, binding agent, lubricating agent, disintegrating agent, colouringagent, flavouring agent or sweetening agent.

In one embodiment, the composition is formulated for selected coated anduncoated tablets, hard and soft gelatine capsules, sugar-coated pills,lozenges, wafer sheets, pellets and powders in a sealed packet.

One embodiment of the invention provides a pharmaceutical compositionconsisting essentially of rifaximin in polymorphic form α.

Another embodiment of the invention provides a pharmaceuticalcomposition consisting essentially of rifaximin in polymorphic form β.

A further embodiment of the invention provides a pharmaceuticalcomposition consisting essentially of rifaximin in polymorphic form γ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a powder X-ray diffractogram of rifaximin polymorphic form α.

FIG. 2 is a powder X-ray diffractogram of rifaximin polymorphic form β.

FIG. 3 is a powder X-ray diffractogram of rifaximin polymorphic form γ.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to the form α, the form β and the form γof the antibiotic known as rifaximin (INN), the processes for theirpreparation and the use thereof in the manufacture of medicinalpreparations for the oral or topical route.

The process of the present invention comprises reacting one molarequivalent of rifamycin O with an excess of 2-amino-4-methylpyridine,preferably from 2.0 to 3.5 molar equivalents, in a solvent mixtureconsisting of water and ethyl alcohol in volumetric ratios between 1:1and 2:1, for a time between 2 and 8 hours at a temperature between 40°C. and 60° C.

After completion of the reaction, the reaction mass is cooled to roomtemperature and added with a solution of ascorbic acid in a mixture ofwater, ethyl alcohol and aqueous concentrated hydrochloric acid, understrong stifling, in order to reduce the small amount of oxidizedrifaximin that forms during the reaction. Finally the pH is adjusted toabout 2.0 by further addition of hydrochloric acid concentrated aqueoussolution, in order to better remove the excess of2-amino-4-methylpyridine used in the reaction. The suspension isfiltered and the resulting solid is washed with the same solvent mixturewater/ethyl alcohol as used in the reaction. Such semifinished productis called “raw rifaximin”.

The raw rifaximin can be directly submitted to the subsequentpurification step. Alternately, in case long times of preservation ofthe semifinished product are expected, the raw rifaximin can be driedunder vacuum at a temperature lower than 65° C. for a time between 6 and24 hours, such semifinished product is called “dried raw rifaximin”.

The resulting raw rifaximin and/or dried raw rifaximin are purified bydissolution in ethyl alcohol at a temperature between 45° C. and 65° C.,followed by crystallization by addition of water, preferably in weightamounts between 15% and 70% to the weight amount of ethyl alcohol usedfor the dissolution, and by keeping the resulting suspension at atemperature between 50° C. and 0° C. under stirring during a timebetween 4 and 36 hours.

The suspension is filtered and the obtained solid is washed with waterand dried under vacuum or under normal pressure, optionally in thepresence of a drying agent, at a temperature between room temperatureand 105° C. for a time between 2 and 72 hours.

The achievement of the α, β and γ forms depends on the conditionsselected for the crystallization. In particular, the composition of thesolvent mixture used for the crystallization, the temperature at whichthe reaction mixture is kept after the crystallization and the period oftime at which that temperature is kept, have proven to be critical.

More precisely, rifaximin γ is obtained when the solution is brought toa temperature between 28° C. and 32° C. to start precipitation and theresulting suspension is further cooled to 0° C. and kept at thistemperature for a time between 6 and 24 hours.

The suspension is filtered, the solid is washed with demineralized waterand is dried to a water content between 1.0% and 2.0%.

The α and β rifaximins are obtained when the temperature is firstbrought to a value between 28° C. and 32° C. in order to startcrystallization, then the suspension is brought to a temperature between40° C. and 50° C. and kept at this value for a time between 6 and 24hours, then the suspension is quickly cooled to 0° C. in 15 minutes toone hour, then is filtered, the solid is washed with water and thendried.

The drying steps plays an important role in obtaining the rifaximin αand β polymorphic forms and has to be monitored by a method suited towater dosage, such as the Karl Fischer method, in order to check theamount of remaining water present in the product under drying.

Rifaximin α or rifaximin β are obtained by drying to different finalwater contents, be they higher or lower than 4.5%, and do not depend onthe experimental conditions of pressure and temperature at which suchcritical water contents are achieved. In fact, the two polymorphicforms, with higher or lower water content, can be obtained by dryingunder vacuum or at atmospheric pressure, at room temperature or at hightemperatures, optionally in the presence of drying agents, provided thatthe drying is prolonged for the time necessary to reach the watercontent characteristic for each polymorphic form.

The polymorphic form β is obtained when the drying of the productcrystallized and washed with water is stopped at water contents higherthan 4.5%, measured by Karl Fischer, preferably between 5.0% and 6.0%,while the polymorphic form α is obtained when drying is continued untilwater contents lower than 4.5%, preferably between 2.0% and 3.0%.

Both the form γ and the forms α and β of rifaximin are hygroscopic, theyreversibly absorb water in time in the presence of suitableenvironmental conditions of pressure and humidity and are susceptible oftransformation from one form into another.

When the polymorphic form α is kept under conditions of relativehumidity higher than 50% for a time between 12 and 48 hours, it changesinto the polymorphic form β, which in its turn is transformed into thepolymorphic form α upon drying to a water content lower than 4.5%,preferably comprised between 2.0% and 3.0%.

Another type of transition exists between the form γ and the forms α andβ, depending upon the temperatures kept during the phase ofprecipitation of rifaximin.

In particular, the form γ turns into the forms α or β when a suspensionof the form γ of rifaximin is kept in an ethyl alcohol/water 7:3 (V/V)solvent mixture at a temperature between 38° C. and 50° C. under strongstifling for a prolonged time, preferably comprised between 6 and 36hours.

After filtration and washing with demineralized water, drying to a watercontent higher than 4.5%, preferably between 5.0% and 6.0%, affords thepolymorphic form β, while when drying is continued to a water contentlower than 4.5%, preferably between 2.0% and 3.0%, gives the form α.

Rifaximins α and β can in their turn change into rifaximin γ bydissolution in ethyl alcohol and treatment of the resulting solution aspreviously described for the preparation of the form γ.

These transitions from one form into another are very important for theinvention, as they can be provide an alternative process for thepreparation of the form desired for the production of the medicinalpreparations. Therefore, the process that allows to transform rifaximinγ into rifaximin α or β in a valid industrial manner, or vice versarifaximin β into rifaximin α, are important parts of the invention.

The process concerning the transformation of rifaximin γ into rifaximinα or rifaximin β comprises suspending rifaximin γ in a solvent mixtureconsisting of ethyl alcohol/water in 7:3 volumetric ratio, warming thesuspension to a temperature between 38° C. and 50° C. and keeping it atthis temperature under strong stifling for a time between 6 and 36hours. The suspension is then filtered, the solid is washed with waterand dried; the polymorphic form β is obtained when drying is carried outto a water content between 5.0% and 6.0% measured by the Karl Fischermethod, while the polymorphic form α is obtained when drying iscontinued to a water content between 2.0% and 3.0%.

The process for the preparation of the form γ starting from rifaximin αor β comprises dissolving the α or β form in ethyl alcohol understifling, at a temperature between 50° C. and 60° C., addingdemineralized water to an ethyl alcohol/water 7:3 volumetric ratio,cooling the solution to 30° C. under strong stifling, cooling theprecipitate to 0° C. and keeping the suspension under stirring at 0° C.for a time between 6 and 24 hours. The suspension is then filtered, thesolid is washed with water and dried to a water content lower than 2.0%thereby obtaining rifaximin γ.

The process for the transformation of the form α into the form βconsists in keeping powder rifaximin α in an ambient having relativehumidity higher than 50% for the time required to obtain a water contentin the powder higher than 4.5%, which time is usually between 12 and 48hours.

The process for the transformation of the form β into the form αconsists in drying powder rifaximin β under vacuum or under conditionsof normal pressure, optionally in the presence of a drying agent, at atemperature between the room temperature and 105° C., for a time between2 and 72 hours, to obtain a water content in the powder lower than 4.5%,preferably between 2.0% and 3.0%.

It is evident from what stated above that during preservation of theproduct particular care should be taken so that the ambient conditionsdo not affect the water content of the product, by preserving theproduct in an environment having controlled humidity or in closedcontainers that allow no significant exchanges of water with theexterior.

The rifaximin α polymorph is characterized by a water content lower than4.5%, preferably between 2.0% and 3.0% and by a powder X-raydiffractogram (reported in FIG. 1) which shows peaks at the values ofthe diffraction angles 2θ of 6.6°; 7.4°; 7.9°; 8.8°; 10.5°; 11.1°;11.8°; 12.9°; 17.6°; 18.5°; 19.7°; 21.0°; 21.4°; 22.1°. The rifaximin βpolymorph is characterized by a water content higher than 4.5%,preferably between 5.0% and 6.0%, and by a powder X-ray diffractogram(reported in FIG. 2) which shows peaks at the values of the diffractionangles 2θ of 5.4°; 6.4°; 7.0°; 7.8°; 9.0°; 10.4°; 13.1°; 14.4°; 17.1°;17.9°; 18.3°; 20.9°.

The rifaximin γ polymorph is characterized by a powder X-raydiffractogram much poorer because of the poor crystallinity; thesignificant peaks are at the values of the diffraction angles 2θ of5.0°; 7.1°; 8.4° as reported in FIG. 3.

The diffractograms have been carried out using a Philips X′Pertinstrument fitted with Bragg-Brentano geometry and under the followingworking conditions:

X-ray tube: Copper

Radiation used: K (α1), K (α2)

Tension and current of the generator: KV 40, mA 40

Monocromator: Graphite

Step size: 0.02

Time per step: 1.25 seconds

Starting and final angular 28 value: 3.0°÷30.0°

The evaluation of the water content in the analyzed samples has alwaysbeen carried out by means of the Karl Fischer method.

Rifaximin α, rifaximin β and rifaximin γ significantly differ from eachfrom other also in terms of bioavailability and intrinsic dissolution.

A bioavailability study of the three polymorphs has been carried out onBeagle female dogs, by feeding them orally with a dose of 100 mg/kg ofone of the polymorphs, collecting blood samples from the jugular vein ofeach animal before each dosing and 1, 2, 4, 6, 8 and 24 hours after eachdosing, transferring the samples into tubes containing heparin andseparating the plasma by centrifugation.

The plasma has been assayed for rifaximin on the validated LC-MS/MS(Liquid Chromathography-Mass Spectrometry/Mass Spectrometry) method andthe maximum plasma concentration observed (C_(max)), the time to reachthe (C_(max)) (t_(max)), and the area under the concentration—time curve(AUC) have been calculated.

The experimental data reported in the following table 1 clearly showthat rifaximin α and rifaximin β are negligibly absorbed, whilerifaximin γ is absorbed at a value (C_(max)=0.668 μg/ml) comprised inthe range of from 0.1 to 1.0 μg/ml.

TABLE 1 Pharmacokinetic parameters for rifaximin polymorphs followingsingle oral administration of 100 mg/kg by capsules to female dogs.C_(max) t_(max) AUC₀₋₂₄ ng/ml h ng · h/ml Mean Mean Mean Polimorph α2.632 9.5 13 Polimorph β 1.096 4 11 Polimorph γ 668.22 2.25 3908

Intrinsic dissolution tests have been carried out on each of the threepolymorphs according to the method described in the monograph 1087 atpages 2512-2513 of the USP (U.S. Pharmacopoeia) 27, clearly showingsignificant differences among rifaximin α, rifaximin β and rifaximin γ.

A sample of each rifaximin polymorph has been put into a die andcompressed at 5 tons by means of a punch of a hydraulic press to obtaina compacted pellet.

The die-holder containing the compacted pellet has then been mounted ona laboratory stirring device, immersed in a dissolution medium androtated by means of the stirring device.

The test, carried out in a dissolution medium made of aqueous phosphatebuffer at ph 7.4 and of sodium lauryl sulfate at a temperature of37±0.5° C., has shown significant differences among the instrinsicdissolution rates exhibited by the three polymorphs.

Rifaximin α has shown disintegration of the compacted pellet within 10minutes so that it has not been possible to calculate the value of itsintrinsic dissolution, while the intrinsic dissolution of rifaximin γhas been about ten times as much that of rifaximin β in accordance withits bioavailability which is more than hundred times as much that ofrifaximin β.

The above experimental results further point out the differencesexisting among the three rifaximin polymorphs.

The forms α, β and γ can be advantageously used in the production ofmedicinal preparations having antibiotic activity, containing rifaximin,for both oral and topical use. The medicinal preparations for oral usewill contain rifaximin α or β or γ together with the usual excipients,for example diluting agents such as mannitol, lactose and sorbitol;binding agents such as starchs, gelatines, sugars, cellulosederivatives, natural gums and polyvinylpyrrolidone; lubricating agentssuch as talc, stearates, hydrogenated vegetable oils, polyethylenglycoland colloidal silicon dioxide; disintegrating agents such as starchs,celluloses, alginates, gums and reticulated polymers; coloring,flavoring and sweetening agents.

The present invention relates to all of the solid preparationsadministrable by the oral route, for instance coated and uncoatedtablets, of soft and hard gelatine capsules, sugar-coated pills,lozenges, wafer sheets, pellets and powders in sealed packets.

The medicinal preparations for topical use will contain rifaximin α or βor γ together with usual excipients, such as white petrolatum, whitewax, lanoline and derivatives thereof, stearylic alcohol, propyleneglycol, sodium lauryl sulfate, ethers of fatty polyoxyethylene alcohols,esters of fatty polyoxyethylene acids, sorbitan monostearate, glycerylmonostearate, propylene glycol monostearate, polyethylene glycols,methylcellulose, hydroxy propylmethylcellulose, sodiumcarboxymethylcellulose, colloidal aluminium and magnesium silicate,sodium alginate.

The present invention relates to all of the topical preparations, forinstance ointments, pomades, creams, gels and lotions.

The invention is further illustrated by some examples. Such examples arenot to be taken as a limitation of the invention, it is in fact evidentthat the α, β and γ forms can be obtained by suitably combining betweenthem the above mentioned conditions of crystallization and drying.

Example 1 Preparation of Raw Rifaximin and of Dried Raw Rifaximin

In a three-necked flask equipped with mechanic stirrer, thermometer andreflux condenser, 120 ml of demineralized water, 96 ml of ethyl alcohol,63.5 g of rifamycin O and 27.2 g of 2-amino-4-methylpyridine are loadedin succession at room temperature. After loading, the mass is heated at47±3° C. and kept under stirring at this temperature for 5 hours, thenis cooled to 20±3° C. and, during 30 minutes, is added with a mixture,prepared separately, of 9 ml of demineralized water, 12.6 ml of ethylalcohol, 1.68 g of ascorbic acid and 9.28 g of aqueous concentratedhydrochloric acid. After completion of the addition, the mass is keptunder stirring for 30 minutes at an inner temperature of 20±3° C. then7.72 g of concentrated hydrochloric acid are dripped until a pH equal to2.0, while keeping said temperature.

After completion of the addition, the mass is kept under stifling for 30minutes, keeping an inner temperature of 20° C., then the precipitate isfiltered and washed with a mixture of 32 ml of demineralized water andof 25 ml of ethyl alcohol. The resulting “raw rifaximin” (89.2 g) isdried under vacuum at room temperature for 12 hours obtaining 64.4 g of“dried raw rifaximin” which shows a water content of 5.6% and adiffractogram corresponding to the polymorphic form β. The product isfurther dried under vacuum until constant weight to afford 62.2 g ofdried raw rifaximin having a water content of 2.2%, whose diffractogramcorresponds to the polymorphic form α.

The product is hygroscopic and the obtained polymorphic form isreversible: the polymorphic form α absorbs water from atmospherichumidity, depending on the relative humidity and the exposure time. Whenthe water content absorbed by the polymorphic form α becomes higher than4.5%, polymorphous α turns to polymorphous β. This in its turn losespart of water by drying, changing into the polymorphic form α when awater content between 2.0% and 3.0% is reached.

Example 2 Preparation of Rifaximin γ

163 ml of ethyl alcohol and 62.2 g of dried raw rifaximin are loaded atroom temperature into a three-necked flask equipped with mechanicstirrer, thermometer and reflux condenser. The suspension is heated at57±3° C. under stifling until complete dissolution of the solid, andadded with 70 ml of demineralized water at this temperature in 30minutes. After completion of the addition the temperature is brought to30° C. in 40 minutes and kept at this value until completecrystallization, then the temperature is further lowered to 0° C. in 2hours and kept at this value for 6 hours. The suspension is thenfiltered and the solid is washed with 180 g of demineralized water anddried under vacuum at room temperature until constant weight, therebyobtaining 52.7 g of pure rifaximin γ having water content of 1.5%.

The form γ is characterized by a powder X-ray diffractogram showingsignificant peaks at diffraction angles 2θ of 5.0°; 7.1°; 8.4°.

Example 3 Preparation of Rifaximin α

62.2 Grams of dried raw rifaximin and 163 ml of ethyl alcohol are loadedat room temperature into a three-necked flask equipped with mechanicstirrer, thermometer and reflux condenser. The suspension is heated at57±3° C. until complete dissolution of the solid and then 70 ml ofdemineralized water are added at this temperature during 30 minutes.After completion of the addition, the temperature is brought to 30° C.during 40 minutes and is kept at this value until plentifulcrystallization. The temperature of the suspension is then brought toabout 40° C. and kept at this value during 20 hours under stifling; thenthe temperature is decreased to 0° C. in 30 minutes and the suspensionis immediately filtered. The solid is washed with 180 ml ofdemineralized water and dried under vacuum at room temperature untilconstant weight, thereby obtaining 51.9 g of rifaximin form α areobtained with a water content equal to 2.5% and a powder X-raydiffractogram showing peaks at values of angles 2θ of 6.6°; 7.4°; 7.9°;8.8°; 10.5°; 11.1°; 11.8°; 12.9°; 17.6°; 18.5°; 19.7°; 21.0°; 21.4°;22.1°.

Example 4 Preparation of Rifaximin α

89.2 Grams of raw rifaximin and 170 ml of ethyl alcohol are loaded atroom temperature into a three-necked flask equipped with mechanicstirrer, thermometer and reflux condenser, then the suspension is heatedat 57±3° C. until complete dissolution of the solid. The temperature isbrought to 50° C. and then 51.7 ml of demineralized water are added atthis temperature during 30 minutes. After completion of the addition thetemperature is brought to 30° C. in one hour and the suspension is keptfor 30 minutes at this temperature obtaining a plentifulcrystallization. The temperature of the suspension is brought to 40° C.and kept at this value during 20 hours under stirring and then furtherlowered to 0° C. during 30 minutes after which the suspension isimmediately filtered. The solid is washed with 240 ml of demineralizedwater and dried under vacuum at 65° C. until constant weight therebyobtaining 46.7 g of rifaximin α with a water content equal to 2.5%.

Example 5 Preparation of Rifaximin α

Example 3 is repeated, but increasing to 50° C. the temperature at whichthe suspension is kept and lowering to 7 hours the time in which thesuspension is kept at this temperature. The product obtained is equal tothat of example 3.

Example 6 Preparation of Rifaximin β

The crystallization of the dried raw rifaximin is carried out accordingto the process described in example 3. Drying under vacuum at roomtemperature is monitored by Karl Fischer and stopped when the watercontent reaches 5.0%: 52.6 g of rifaximin β are obtained characterizedby a powder X-ray diffractogram showing peaks at values of angles 2θ of5.4°; 6.4°; 7.0°; 7.8°; 9.0°; 10.4°; 13.1°, 14.4°; 17.1°; 17.9°; 18.3°;20.9°.

Example 7

Preparation of Rifaximin α Starting from Rifaximin γ

Grams of rifaximin γ are suspended in a mixture of 13 ml of ethylalcohol and 5.6 ml of water and the suspension is heated at 40° C.during 24 hours under stirring in a 50 ml flask equipped with condenser,thermometer and mechanic stirrer. The suspension is then filtered andthe solid is washed with water, then dried under vacuum at roomtemperature until constant weight. 4 Grams of rifaximin are obtainedshowing a powder X-ray diffractogram corresponding to that of thepolymorphic form α and a water content equal to 2.6%.

Example 8

Preparation of Rifaximin γ Starting from Rifaximin α

Grams of rifaximin form α and 52.4 ml of ethyl alcohol are loaded into a250 ml three-necked flask equipped with reflux condenser, thermometerand mechanical stirrer; the suspension is heated under stirring at thetemperature of 50° C. until complete dissolution of the solid.

The clear solution is added with 22.5 ml of water in 30 minutes understirring, cooled to 30° C. and kept at this temperature for 30 minutes.The formed suspension is cooled to 0° C. under strong stirring and keptat this temperature during 6 hours. After this time, part of thesuspension is taken, filtered, washed with demineralized water and driedunder vacuum at 30° C. until constant weight.

The resulting product, 3.7 g, shows a diffractogram consistent with thatof the form 7 and a water content of 1.7%.

The remaining part of the suspension is kept at 0° C. for further 18hours under strong stirring and then is filtered, washed withdemineralized water and dried at 30° C. under vacuum until constantweight. 9 Grams of product showing a diffractogram consistent with thatof the form γ and a water content equal to 1.6% are obtained.

Example 9

Preparation of Rifaximin α Starting from Rifaximin β

Grams of rifaximin β having a water content equal to 5.0% are driedunder vacuum at +30° C. during 8 hours obtaining 4.85 g of rifaximin αhaving a water content equal to 2.3%.

Example 10

Preparation of Rifaximin β Starting from Rifaximin α

Grams of rifaximin α having a water content equal to 2.5% are keptduring 40 hours in an atmosphere containing a relative humidity equal to56% made by means of a saturated aqueous solution of calcium nitratetetrahydrate. 5.17 Grams of rifaximin β with a water content equal to5.9% are obtained after this time.

Example 11 Bioavailability in Dogs by Oral Route

Twelve 20 week pure-bred Beagle females dogs, and weighing between 5.0and 7.5 kg, have been divided into three groups of four.

The first of these three groups has been treated with rifaximin α, thesecond with rifaximin β and third with rifaximin γ according to thefollowing procedure.

Each dog received orally 100 mg/kg of one of the rifaximin polymorphs ingelatin capsules and 2 ml blood samples were collected from the jugularvein of each animal before each administration and 1, 2, 4, 6, 8 and 24hours after the administration. Each sample was transferred into anheparinized tube and was centrifuged; the plasma was divided into 500two aliquots and frozen at −20° C.

The rifaximin contained in the plasma was assayed by means of thevalidated LC-MS/MS method and the following parameters were calculatedaccording to standard non-compartmental analysis:

C_(max)=maximum plasma concentration of rifaximin observed in theplasma;

T_(max)=time at which the C_(max) is reached;

AUC=area under the concentration-time curve calculated through thelinear trapezoidal rule.

The results reported in the following table 2 clearly show how therifaximin is very much more absorbed, more than 102 times, in respect ofrifaximin α and rifaximin β which are practically not absorbed.

TABLE 2 Pharmacokinetic parameters for rifaximin polymorphs followingsingle oral administration of 100 mg/kg by capsules to female dogs.C_(max) t_(max) AUC₀₋₂₄ ng/ml h ng · h/ml Mean Mean Mean rifaximin α2.632 9.5 13 rifaximin β 1.096 4 11 rifaximin γ 668.22 2.25 3908

Example 12 Intrinsic Dissolution Test

A sample of 100 mg of each rifaximin polymorph was submitted to theintrinsic dissolution test carried out as described in the monograph1087 at pages 2512-2513 of the USP (U.S. Pharmacopoeia) 27.

100 Milligrams of a rifaximin polymorph were put into a die andcompressed for 1 minute under a pressure of 5 tons by means of a punchin a hydraulic press.

A compacted pellet was formed in the die with a single face of definedarea exposed on the bottom of the die so that from 50% to 75% of thecompacted pellet could dissolve in an appropriate dissolution medium.

The holder containing the die was mounted on a laboratory stiflingdevice, immersed in a glass vessel containing a dissolution medium androtated at a rotation speed of 100 rpm by means of the stifling device,while keeping the temperature of the dissolution medium at 37±0.5° C.The dissolution medium contained in the glass vessel consisted of 1000ml of 0.1 M aqueous phosphate buffer pH 7.4 containing 4.5 g of sodiumlauryl sulfate and was kept at 37±0.5° C. for the whole duration of thetest.

Samples of 2 ml of solution were taken after 15, 30, 45 and 60 minutesfrom the start of the dissolution procedure and analyzed by HPLC for theamount of rifaximin dissolved.

The sample containing rifaximin α systematically showed disintegrationof the compacted pellet within 10 minutes and said phenomenon was alsopresent at lower concentrations (0.1% and 0.3%) of sodium lauryl sulfateand even in absence of said surfactant, so that the value of itsintrinsic dissolution could not be calculated.

The intrinsic dissolution of rifaximin γ was about ten times as muchthat of rifaximin β at every time, as it can be inferred by theexperimental results shown in the following table 3.

TABLE 3 Intrinsic dissolution in 0.1M aqueous phosphate buffer pH 7.4with 0.45% sodium lauryl sulfate Rifaximin dissolved (mg/cm²) Time (min)β polymorph γ polymorph 15 0.28 2.46 30 0.50 4.52 45 0.72 6.44 60 0.949.04 Intrinsic dissolution 0.0147 0.1444 rate (mg/min/cm²)

1. A pharmaceutical composition comprising rifaximin in polymorphic formα.
 2. A pharmaceutical composition comprising rifaximin in polymorphicform β.
 3. A pharmaceutical composition comprising rifaximin inpolymorphic form γ.